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PLoS BIOLOGY
Structure of a Pheromone Receptor-Associated
MHC Molecule with an Open and Empty Groove
Rich Olson1, Kathryn E. Huey-Tubman1,2, Catherine Dulac3, Pamela J. Bjorkman1,2*
1 Division of Biology, California Institute of Technology, Pasadena, California, United States of America, 2 Howard Hughes Medical Institute, California Institute of Technology,
Pasadena, California, United States of America, 3 Department of Molecular and Cellular Biology, Howard Hughes Medical Institute, Harvard University, Cambridge,
Massachusetts, United States of America
Neurons in the murine vomeronasal organ (VNO) express a family of class Ib major histocompatibility complex (MHC)
proteins (M10s) that interact with the V2R class of VNO receptors. This interaction may play a direct role in the
detection of pheromonal cues that initiate reproductive and territorial behaviors. The crystal structure of M10.5, an
M10 family member, is similar to that of classical MHC molecules. However, the M10.5 counterpart of the MHC peptidebinding groove is open and unoccupied, revealing the first structure of an empty class I MHC molecule. Similar to
empty MHC molecules, but unlike peptide-filled MHC proteins and non-peptide–binding MHC homologs, M10.5 is
thermally unstable, suggesting that its groove is normally occupied. However, M10.5 does not bind endogenous
peptides when expressed in mammalian cells or when offered a mixture of class I–binding peptides. The F pocket side
of the M10.5 groove is open, suggesting that ligands larger than 8–10-mer class I–binding peptides could fit by
extending out of the groove. Moreover, variable residues point up from the groove helices, rather than toward the
groove as in classical MHC structures. These data suggest that M10s are unlikely to provide specific recognition of class
I MHC–binding peptides, but are consistent with binding to other ligands, including proteins such as the V2Rs.
Citation: Olson R, Huey-Tubman KE, Dulac C, Bjorkman PJ (2005) Structure of a pheromone receptor-associated MHC molecule with an open and empty groove. PLoS Biol
3(8): e257.
Recently, it was shown that the V2R class of pheromone
receptors specifically interacts with members of the mouse
M1 and M10 families [7] of major histocompatibility
complex (MHC) class Ib proteins [8], which do not appear
to have human orthologs [9]. Classical class I MHC
molecules, which exhibit high polymorphism in mice,
humans, and other mammals, present peptides derived from
cytoplasmic proteins to T cells during immune surveillance,
and are expressed on most or all nucleated cells [10]. The
less polymorphic non-classical class Ib molecules are expressed on a more limited subset of cells and are involved in
a variety of functions, including presentation of hydrophobic peptides (e.g., by Qa-2), presentation of formylated
peptides by H2-M3, and lipid presentation by CD1 proteins
[11]. The non-classical M1 and M10 proteins are expressed
exclusively in the VNO and appear to facilitate cell surface
expression of V2Rs. Male mice that are genetically deficient
in the class I MHC–associated b2-microglobulin (b2m) light
chain show no surface expression of V2R pheromone
receptors in the dendritic terminals of VNO sensory
Introduction
In most mammals, chemical communication between
conspecific animals is involved in initiation of reproductive
and territorial behaviors. The detection of these species- and
gender-specific chemical cues, also called pheromones, is
thought to involve receptors of the vomeronasal organ (VNO),
a small neuronal epithelium located between the nasal cavity
and the palate [1]. Although neurons of the main olfactory
epithelium involved in odorant detection ultimately project
to cognitive regions of the brain, vomeronasal neurons send
inputs via the accessory olfactory bulb to specialized centers
of the hypothalamus and amygdala, where they elicit neuroendocrine responses and behaviors such as oestrous synchronization, aggression, and sex discrimination [1–3].
Pheromone receptors belong to the ubiquitous family of G
protein–coupled receptors (GPCRs), but are unrelated in
sequence to main olfactory epithelium receptors that detect
volatile odorants [4,5]. Mouse pheromone receptors can be
divided into two subtypes, V1R and V2R, each of which is
expressed in the dendritic tips of bipolar neurons in spatially
distinct regions of the VNO. The human orthologs of most of
these genes appear to be pseudogenes [1]. Mouse V1R
receptors are found in the apical VNO domain, are thought
to signal through the G-protein a-subunit Gai2, and exhibit
sequence similarity to the T2R family of bitter taste receptors
[6]. V2R receptors, in contrast, are found in the basal VNO
domain, are likely to signal via the Gao molecule, and are
related in sequence to metabotropic glutamate (mGluRs),
GABAB (c-aminobutyric acid-B), and calcium sensing receptors. V1R and V2R receptor family members, like all G
protein–coupled receptors, contain seven putative transmembrane helices, but, in addition, V2R members include a
large N-terminal extracellular domain.
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Received April 6, 2005; Accepted May 18, 2005; Published July 12, 2005
DOI: 10.1371/journal.pbio.0030257
Copyright: Ó 2005 Olson et al. This is an open-access article distributed under the
terms of the Creative Commons Attribution License, which permits unrestricted
use, distribution, and reproduction in any medium, provided the original work is
properly cited.
Abbreviations: b2m, b2-microglobulin; CD, circular dichroism; CHO, Chinese
hamster ovary; ER, endoplasmic reticulum; MALDI-TOF, matrix-assisted laser
desorption ionization time-of-flight; MHC, major histocompatibility complex;
MUPs, major urinary proteins; NCS, non-crystallographic symmetry; VNO, vomeronasal organ
Academic Editor: Andrej Sali, University of California, San Francisco, United States
of America
*To whom correspondence should be addressed. E-mail: [email protected]
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August 2005 | Volume 3 | Issue 8 | e257
Crystal Structure of the MHC Ib Molecule M10.5
neurons and lack aggressive behavior toward intruder males
[8]. Facilitation of V2R surface expression appears to involve
M10 binding to a V2R because immunopurification of VNO
receptors identified a multimolecular complex formed
between M10, b2m, and a V2R protein. A given neuron
generally expresses only one of the roughly 150 different
subtypes of V2R receptor and one of the nine known VNO–
specific MHC class Ib proteins (six M10 and three M1 family
genes) [8,12]. Genetic analysis of individual neurons suggests
specificity in binding between different V2R and M10
proteins [8].
Homologous receptors to the V2R proteins, such as the
GABAB receptors and the umami and sweet taste receptors,
also require accessory proteins for effective surface expression [13–15]. However, the nature of M10s as MHC molecules
together with the association between the MHC and mating
behavior in rodents [16] offers the intriguing possibility that
M10 proteins play a direct role in mate or species
recognition. Mice tend to choose mates with disparate MHC
haplotypes [17], and pregnant mice have a higher incidence
of spontaneous abortion if exposed to the scent of a male
with a different MHC haplotype (the Bruce effect) [18]. These
functions, as well as other species-specific indicators, may be
mediated via M10-V2R complexes, perhaps by binding
peptides or other accessory molecules in the canonical
peptide-binding groove that exists in classical class I MHC
structures [10]. Peptide binding during heavy and light chain
assembly in the endoplasmic reticulum (ER), as occurs for
classical class I MHC molecules, seems unlikely for M10
proteins because in situ hybridization assays demonstrate
that TAP1 and TAP2, which are required for transport of
cytoplasmic-derived peptides into the ER, are not expressed
in the VNO [8]. However, a recent study shows that V2R–
containing neurons are activated by nonameric class I MHC–
binding peptides [19], suggesting involvement of exogenously
acquired MHC–binding peptides, and perhaps M10 proteins,
in individual mate or species recognition.
To determine the peptide-binding capability and structural
characteristics of V2R receptor-associated MHC molecules,
we expressed, characterized, and solved the crystal structure
of the ectodomain of M10.5, an M10 family member. The
M10.5 structure reveals an open conformation of the a1–a2
domain helices that contains no ordered peptidic or nonpeptidic occupant, which represents the first example, to our
knowledge, of the structure of an empty class I MHC
molecule. Thermal stability studies suggest that the M10.5
groove is normally occupied; however M10.5 does not
associate with the sorts of peptides that normally bind to
class I MHC molecules. These results suggest a new and
divergent function for the M10 family of proteins.
heterodimeric complexes with human, as compared with
mouse, b2m [20,21]. M10.5 was crystallized in space group
P212121 with five molecules in the asymmetric unit. Most
crystals diffracted weakly to 3.5–4.0 Å resolution, but an
incomplete dataset from a rare crystal that diffracted beyond
3.5 Å was combined with a more complete dataset to 4.0 Å
(Table 1). The structure was solved by molecular replacement
using the mouse class Ib MHC molecule Qa-2 [22] as a search
model. Refinement using non-crystallographic symmetry
(NCS) restraints yielded a final model (Rcryst ¼ 26.6%; Rfree
¼ 30.3%) (Table 1). Although data to 3.0 Å were included in
the refinement, the high-resolution data are incomplete, thus
the effective resolution of the structure is 3.2 Å. Two loops
comprising residues 145–150 (a2 domain) and 194–197 (a3
domain) are missing in electron-density maps and are not
included in our model.
The overall structure of M10.5 resembles the structures of
classical class I MHC molecules. A BLAST search [23]
identifies mouse H-2Dd as the most closely related classical
class I MHC molecule (approximately 50% amino acid
identity) for which a structure is available [24], thus we have
used H-2Dd for comparisons. As in other class I structures,
the first 180 residues of the M10.5 heavy chain form the a1–a2
domain superdomain, which is composed of an eightstranded anti-parallel b-sheet platform topped by two antiparallel a-helices. The following approximately 90 residues
form the a3 domain, which resembles an immunoglobulin
constant region domain (Figure 1A and 1B). The non-
Table 1. Crystallographic Data and Refinement Statistics
Category
Characteristic
Strict NCS
Data collection
Wavelength (Å)
Resolution (Å)
Unique reflections
Total reflections
Mean redundancy
Completeness (%)
Rmergea (%)
I/rI
1.00879,1.11587
100–3.0 (3.31–3.2)
40,549
262,067
6.5 (5.2)
95.5 (83.9)
15.4 (43.7)
9.2 (3.2)
Resolution (Å)
Reflections in working set
Reflections in test set
Rcrystc (%)
Rfreec (%)
Atoms (#)
RMS deviations from
ideality
RMS deviations bonds (Å)
RMS deviations angles (8)
Ramachandran plot (%)
Most favored
Additionally allowed
Generously allowed
Disallowed
50–3.0b
44,543
2,355
30.9
30.7
2,733
50–3.0b
44,543
2,355
26.6
30.3
13,720
0.009
1.84
0.008
1.72
83.8
14.6
1.3
0.3
83.5
13.7
2.4
0.4
Refinement
statistics
Results
The M10.5 Structure
A soluble form of M10.5 was expressed together with
human b2m in baculovirus-infected insect cells, and soluble
M10.5–b2m complexes were purified from cell supernatants.
Similar efforts to express soluble M10.5 together with mouse
b2m in insect cells or Chinese hamster ovary (CHO) cells were
unsuccessful (data not shown), perhaps related to the
observation that mouse class I MHC molecules form stronger
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Restrained
NCS
-
a
Rmerge (I) ¼ RhkljIi,Ih.j/RhklIi, where Ii is the i-th observation of the intensity of unique reflection hkl and ,Ih. is
the mean intensity of reflection hkl. Numbers in parenthesis indicate the highest resolution shell.
Reflections between 3.2 and 3.0 Å resolution were included in the refinement, but due to insufficient
completeness, we are reporting this as a 3.2 Å structure.
c
Rcryst (F) ¼ RhkljjFobsj – jFcalcjj/RhkljFobsj, where jFobsj and jFcalcj are the observed and calculated structure factor
amplitudes, respectively. Rfree is the crystallographic R-factor calculated for 5% of the data withheld from refinement.
RMS, root mean square.
DOI: 10.1371/journal.pbio.0030257.t001
b
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Crystal Structure of the MHC Ib Molecule M10.5
Figure 1. The Structure of M10.5
(A) Ribbon diagram of M10.5 (side view). The heavy chain is blue, the b2m light chain is green, disulfide bonds are yellow, and ordered carbohydrate
attached to Asn223 is shown in ball-and-stick representation. Two disordered loops in the heavy chain are shown as dashed blue lines.
(B) Top view of the a1–a2 platform overlaid with an Fo-Fc annealed omit electron-density map contoured at 3.5 r. The map was calculated for one of
five molecules in the asymmetric unit using NCS restraints in the annealing process. Residues 55–84 and 137–174, shown in stick representation, were
omitted from the structure factor calculation. Electron density is absent for residues 145–150 (dashed line), indicating that they are disordered. No
significant electron density is observed in the groove between the a1 and a2 helices.
(C) Stereo view of the superposition of the a1–a2 platforms from M10.5, H-2Dd [24], FcRn [32], and HFE [33]. Structures were aligned using residues
classified as platform b-sheet residues (see Materials and Methods). The cleft between a1 and a2 helices is significantly narrower in FcRn and HFE than
in M10.5 and H-2Dd.
DOI: 10.1371/journal.pbio.0030257.g001
covalently attached b2m light chain, also resembling an
immunoglobulin constant region, contacts both the underside of the a1–a2 platform and one b-sheet of the a3 domain
in an orientation consistent with previously solved mouse and
human class I MHC structures, indicating that pairing human
b2m with the mouse M10.5 heavy chain does not disrupt the
overall M10.5 structure.
Ordered N-linked carbohydrate is observed attached to
Asn223 within the loop joining the third and fourth b-strand
in the a3 domain. The carbohydrate occludes the counterpart
of the region in class I MHC molecules that is the major
determinant for binding the T cell co-receptor CD8 [25],
likely preventing M10.5 from participating in CD8þ T cell–
mediated immunological responses. Ordered carbohydrate is
not observed at either of the two other predicted N-linked
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glycosylation sites (Asn62 and Asn198), but the quality of the
electron-density map is poor in these regions.
The a1–a2 Platform of M10.5 Contains an Open, but
Apparently Empty, Groove
A large groove between the a1 and a2 domain helices forms
the peptide-binding site in classical class I MHC molecules
[10]. Structural studies of class I MHC molecules and
homologs show a correlation between the degree of separation of the a1–a2 domain helices and the ability to bind
peptides or other small molecules [26]. The helices are
separated by approximately 18 Å in the center of the grooves
of classical peptide-binding class I MHC molecules such as H2Dd [24], and class I MHC-related proteins that bind other
small molecule ligands also contain open grooves with
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Crystal Structure of the MHC Ib Molecule M10.5
separated a1 and a2 domain helices [27–31]. In contrast, the
neonatal Fc receptor (FcRn) [32], the hemochromatosis
protein HFE [33], and MIC-A [34], MHC homologs that do
not bind small molecule ligands, have collapsed grooves with a
smaller separation between the a1–a2 domain helices [32–34].
A superposition of the a1–a2 platforms of M10.5, H-2Dd,
FcRn, and HFE illustrates the variation in groove size (Figure
1C) and demonstrates that M10.5 has an open groove more
similar to the peptide-binding classical class I MHC molecules
than the non–peptide-binding homologs. The M10.5 a1–a2
domain helices superimpose well with the analogous H-2Dd
helices (RMS [root mean square] deviation of 1.22 Å for 164
of 181 a-carbon atoms), with the largest differences located in
the region of the a2 domain helix immediately preceding six
residues that are disordered in M10.5 (residues 145 to 150).
The overall similarity of the M10.5 and H-2Dd a1–a2
platforms, which are contained within class I heavy chains
that are paired with human (M10.5) or mouse (H-2Dd) b2m
light chains, demonstrates that the observed structure of the
M10.5 a1–a2 platform does not result from an artifactual
change induced by pairing with human, rather than mouse,
b2m. The more open character of the M10.5 groove as
compared with the grooves of non–peptide-binding MHC
homologs is seen when the calculated accessible surface areas
are compared: approximately 730 Å2 for M10.5 compared
with approximately 760 Å2 for typical class I MHC molecules
[30,33], approximately 690 Å2 for H-2Dd, and approximately
415 Å2 and approximately 235 Å2 for HFE and FcRn,
respectively [33] (see Materials and Methods). Thus the
M10.5 groove can be classified as ‘‘open’’ and capable of
binding to a ligand.
The a1–a2 domain groove is occupied by a peptide or
mixture of peptides in all class I MHC structures solved to
date, and continuous electron-density representing peptide(s)
is always seen in the a1–a2 groove [35]. Indeed, attempts to
crystallize an empty version of H-2Kb, which was expressed in
cells lacking peptide-loading machinery and purified in the
absence of a binding peptide, resulted in a crystal structure
that revealed a peptide derived from the cell growth media
[36]. M10.5 omit electron-density maps in which the a1 and
a2 helices were removed from structure factor calculations
return clear density for the omitted region (Figure 1B),
indicating that the maps are of sufficient quality to detect
bound molecules of the size of an 8–10 residue peptide.
However, M10.5 electron-density maps show no ordered
density corresponding to a peptide: Annealed Fo-Fc maps
calculated using NCS constraints or tight restraints do not
show continuous electron density within the groove (Figure
S1), and maps calculated before or after 5-fold real space
averaging also failed to reveal unbroken density in the cleft
(data not shown). The possibility that a minor peak near the
center of the groove in averaged maps represents a portion of
a bound polyethylene glycol molecule is discussed in the
caption of Figure 1. We conclude that the M10.5 groove does
not contain a single defined peptidic or non-peptidic
occupant or a mixture of compounds with a similar
conformation.
Figure 2. Groove Surface Characteristics
(A) Electrostatic surface representations calculated using GRASP [42] of
the M10.5 (left) and H-2Dd (right) grooves. Positions of the six pockets are
labeled A–F in yellow. In H-2Dd, Arg62 and Glu163 create a bridge over
the peptide-binding groove. In M10.5, Glu63 and Arg167 define a similar
feature. The H-2Dd groove is constricted between Asn70 and Arg155. In
M10.5, Ala70 and Gly155 lead to a wider groove and continuous D and E
pockets.
(B) Comparison of residues in the A and F pockets of classical class I MHC
molecules and M10.5. All alanine-peptide (yellow, atoms color-coded
according to atom type) is derived from the H-2Dd structure [24]. Left: In
class I MHC proteins, a cluster of four tyrosine residues in the A pocket
(Tyr7, Tyr59, Tyr159, and Tyr171) form hydrogen bonds with backbone
atoms in the peptide N-terminus (residues are listed in single letter code
for M10.5 and H-2Dd, respectively). In M10.5, Tyr7 is replaced by a
threonine residue and Tyr171 is replaced with a cysteine. Five of the six
M10 proteins have an additional tyrosine at position 33 that could
potentially replace one of the two missing tyrosine residues. M10.5 has a
serine instead of a glycine at position 26, which could participate in
additional hydrogen-binding interactions. Right: The F pocket of class I
MHC molecules is blocked on one end by Thr80, Tyr84, and Lys146. The
M10.5 pocket is open on this end (see panels A and C) due to the
substitution of a glutamate residue for Tyr84 and the disorder of the loop
containing residue 146 (Asp in M10.5). Lys142 in M10.5 is missing
sidechain density and has been modeled as an alanine, but most likely
would not further occlude the M10.5 F pocket.
(C) Molecular surface representations of M10.5 and H-2Dd with an allalanine peptide (yellow) derived from the H-2Dd structure [24] superimposed in the M10.5 groove. M10.5 atoms that came within 2.5 Å of the
peptide trace were considered clashes and are colored red. Four
additional alanine residues (green) were added to the C-terminus of the
H-2Dd–binding peptide to illustrate that peptides binding in the M10.5
groove could extend out of the F pocket side.
DOI: 10.1371/journal.pbio.0030257.g002
peptides (Figure 2A) [37–39]. The A and F pockets at either
end of the peptide-binding groove are largely conserved and
interact with the N- and C-terminus, respectively, of the
bound peptide [37,39]. The B, C, D, and E pockets contain
residues that vary between alleles, resulting in different
allele-specific peptide-binding preferences.
Comparison of M10.5 and Classical Class I MHC Grooves
Crystallographic studies of classical class I MHC–peptide
complexes have defined six pockets (A–F) within the peptidebinding groove that interact with various portions of bound
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Crystal Structure of the MHC Ib Molecule M10.5
The A and F pocket regions of the M10.5 groove contain
substitutions that prevent the interactions that anchor
peptide termini into the groove of a classical class I MHC
molecule. Two of the four conserved tyrosines in the MHC
class I A pocket (tyrosines 7, 59, 159, and 171), which
hydrogen bond directly or through a water molecule with
main chain atoms of the peptide N-terminal residue [37,39],
are substituted in M10.5 as Thr7 and Cys171 (Figure 2B).
Substitutions in other M10s also eliminate one or two of the
four A pocket tyrosines [8,12]. It remains possible, however,
that M10.5 and other M10s could bind a peptide N-terminus
in a non-classical manner, utilizing residues including Thr7,
Ser26, and Tyr33 as hydrogen-bond donors. Residues that
typically anchor the C-terminus of a peptide in the class I
MHC F pocket are also different in M10.5. In H-2Dd, the
ninth and tenth residue of the bound peptide (P9 and P10)
form hydrogen-bonding and van der Waals contacts with
Val76, Asp77, Tyr84, Thr143, Lys146, and Trp147 (Figure
2B). In M10.5 and most other M10s, these residues are
replaced by non-conservative substitutions, and the last two
are missing entirely from the M10.5 electron-density map.
Thus potential interactions between M10 F pocket groove
residues and peptides would have to occur in a non-classical
manner.
An interesting feature of possible functional relevance is
the relatively open nature of the F pocket side of the M10.5
groove (Figure 2C), suggesting that potential small molecule
or peptide occupants could extend out of this side of the
groove. Homology models of other M10 proteins constructed
using the M10.5 structure and the Swiss-model Protein
modeling Server [40] suggest that this open character is a
general feature of M10 proteins (data not shown). In class I
MHC molecules, Tyr84 and Lys146 occlude the F pocket side
of the groove, but the tyrosine to glutamate substitution at
position 84 in M10.5 (Figure 2B) contributes to the more
open M10.5 groove. In addition, M10.5 Asp146 is part of the
disordered 145–150 region of the a2 domain helix, thus it
does not contribute to closing the F pocket side of the
groove. The disorder of these residues may be related to a
need to maintain flexibility in the F pocket end of the M10.5
groove to allow binding of ligands that extend out of the
M10.5 groove.
Table 2. Groove Residues
Residue
H-2Dd
H-2Dd Pocketa
M10.5
M10.5 Pocketa
Peptide Clash?b
5
7
9
24
26
33
34
52
55
59
63
66
70
74
77
80
81
95
97
99
114
116
122
123
124
133
139
140
143
147
152
156
159
167
171
Leu
Tyr
Val
Glu
Gly
Phe
Val
Ile
Glu
Tyr
Glu
Arg
Asn
Phe
Asp
Thr
Ala
Leu
Trp
Ala
Trp
Phe
Asp
Tyr
Ile
Trp
Ala
Ala
Thr
Trp
Ala
Asp
Tyr
Trpc
Tyr
Buried
A,B
Buried
Buried
Buried
Buried
Buried
Buried
Buried
A
A,B
B
B,C
C
F
F
F
F
C
C,D
D,E
C,E,F
Buried
F
Buried
Buried
Buried
Buried
F
E,F
E
E
A,D
A
A
Leu
Thr
Arg
Gln
Ser
Tyr
Gln
Met
Glu
Tyr
Glu
Hisc
Ala
Ala
Thr
Tyrc
Met
Leu
Glu
Phe
Tyr
Leu
Asp
Tyr
Ile
Trp
Val
Gly
Phec
Arg
Valc
Trp
Tyr
Arg
Cys
A
A,B
C,D
B
A,B
A
A
A
A
A
A,B
B
B,C
C
F
F
F
C
C
B,D
E
C,E,F
F
Buried
E,F
E
F
F
F
Missing
D,E
D
A,D
A
A
No
No
Yes
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
Yes
No
No
No
No
No
No
No
No
No
No
No
No
No
Yes
No
Conserved (blue) or highly variable (red) residues are defined as in Figure 4.
a
Groove residues have 5.0 Å2 or more of accessible surface area to a 1.4 Å radius probe but less than 5.0 Å2 to a 5.0 Å radius probe. ‘‘Buried’’ residues have less than 5.0 Å2 of accessible surface area to a 1.4 Å radius probe. ‘‘Missing’’ residues
are disordered.
b
Residues with atoms less than 2.5 Å from a polyalanine version of an H-2Dd–binding peptide superimposed in the M10.5 groove.
c
Residues that have accessible surface area in the groove but have more than 5.0 Å2 of accessible surface area to a 5.0 Å radius probe.
DOI: 10.1371/journal.pbio.0030257.t002
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Crystal Structure of the MHC Ib Molecule M10.5
Figure 3. Groove Residues in M10.5 and H-2Dd
Stereo view of residues lining the a1–a2 groove in M10.5 (A) and H-2Dd (B). Groove residues are defined as in Table 2.
DOI: 10.1371/journal.pbio.0030257.g003
Asn70 and Arg155 with alanine and glycine residues, respectively, removing the constriction that sandwiches the peptide
residue at position 4 (glycine) in the H-2Dd structure [24].
The chemical character of the groove of MHC–related
protein can sometimes reveal the nature of its ligand. For
example, the largely hydrophobic grooves of CD1 [30] and the
class Ib MHC molecules Qa-2 [22] and HLA-E [41] allow
binding of lipids (CD1) and hydrophobic peptides (the class
Ib proteins). By contrast, the M10.5 groove, like the grooves of
H-2Dd and other classical class I MHC molecules, contains a
mixture of polar and non-polar residues (Table 2). A surface
potential map generated by GRASP [42] shows that the A, C,
E, and F pockets of M10.5 are slightly acidic whereas the B
and D pockets are uncharged (see Figure 2A). The sidechain
of Arg9 (a buried valine in H-2Dd) points into the center of
the M10.5 groove and forms a salt-bridge with the sidechain
of Glu97. A second salt-bridge between Glu63 and Arg167
bridges the A and B pockets. The groove in H-2Dd is
primarily acidic, with a similar salt bridge formed between
Arg62 and Glu163 over the A–B pocket boundary (Figure 2A).
Peptides bound to H-2Dd are accommodated under the salt
bridge [24], thus the salt bridge in the M10.5 groove would
not necessarily prevent binding of a peptide or other small
molecule.
In order to make additional comparisons between the
grooves of M10.5 and peptide-binding class I MHC molecules,
we used a previously described algorithm [30,33] to identify
residues that comprise the grooves of M10.5 and H-2Dd.
Residues that line the M10.5 and H-2Dd grooves were defined as
having more than 5.0 Å2 accessible surface area to a 1.4 Å probe
and less than 5.0 Å2 to a 5.0 Å probe (Table 2, Figure 3A and 3B),
resulting in 33 groove residues in M10.5 and 22 groove residues
in H-2Dd. The additional groove residues in M10.5 that are
buried in H-2Dd are divided into two clusters, one in the area
surrounding the A and B pockets, and the other around the F
pocket (see Figure 2A). The M10.5 A pocket is larger than its H2Dd counterpart due to the substitution of class I MHC residues
Tyr7 and Tyr171 with less-bulky threonine and cysteine
residues, respectively, and a different location of the sidechain
of M10.5 Arg167 as compared to H-2Dd Trp167. Arg167 was
postulated to likely block peptide binding in M10.5 [8] by
analogy to Arg167 in FcRn, which partially occludes the A-B
pocket [32]. However, the sidechain of Arg167 in M10.5 is
partially disordered, suggesting mobility that could result in a
rearrangement to allow a groove occupant to access the open
pocket area below the arginine sidechain. Other differences
that create a larger M10.5 A pocket compared to H-2Dd involve
several large aromatic residues lining the H-2Dd groove that
are replaced by smaller non-aromatic residues in M10.5. These
include H-2Dd Tyr7, Phe74, Trp97, Phe116, Trp167, and
Tyr171, which are substituted as Thr7, Ala74, Glu97, Leu116,
Arg167, and Cys171 in M10.5. Another difference between the
M10.5 and H-2Dd grooves is a considerable enlargement of the
M10.5 D pocket, which is continuous with the E pocket. The
enlargement is caused by the replacement of H-2Dd residues
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Sequence Conservation and Receptor Binding
Like classical class I MHC molecules, variability within M10
proteins is mainly localized to the a1–a2 platform, with the a3
domain being more constant [8,12]. As previously predicted
[8] and now confirmed by the M10.5 structure, amino acids
with the highest degree of sequence variability within the M10
and M1 families cluster on the top face of the a1 and a2
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Crystal Structure of the MHC Ib Molecule M10.5
Figure 4. Sequence Conservation in M10 and H-2 Loci
(A) Ribbon diagram of the M10.5 a1–a2 platform with positions of
residues that are 100% identical in the nine M10 and M1 families colored
blue; residues that are among the top 10% most variable are colored red.
Variability is determined by the number of amino acids at a given
position divided by the frequency of the most common allele at that
position. Residues 145–150, which are disordered, are designated by a
dashed line.
(B) Ribbon diagram of the H-2Dd a1–a2 domain. Conserved (blue) and
highly variable (red) residues are derived from an alignment of 19 H-2D
alleles [80].
DOI: 10.1371/journal.pbio.0030257.g004
Figure 5. Thermal Denaturation Profiles
Thermal denaturation of M10.5 (A) compared with empty and peptidefilled H-2Kd (B) (modified from Figure 3A in Fahnestock et al. [21]). The
CD signal at 223 nm was monitored as a function of temperature for
insect cell-derived M10.5 and empty and peptide-filled versions of
soluble H-2Kd produced in CHO cells. Tms for the melting of the M10.5
heavy chain and b2m light chain (marked with arrows) were derived by
estimating the half-point of the ellipticity change between the beginning
and end of each transition. The M10.5 denaturation profile is similar to
the profile obtained from empty H-2Kd, suggesting that the M10.5
groove is not occupied. The possibility that polyethylene glycol, a
component in the crystallization solutions, might bind to and stabilize
M10.5 was investigated by repeating the unfolding experiment in the
presence of 20% polyethylene glycol 1000, with no significant changes
to the thermal stability profile (data not shown).
DOI: 10.1371/journal.pbio.0030257.g005
domain helices. Indeed, three of the six disordered residues
(145–150) in the M10.5 a2 domain helix belong to the top
10% of variable residues within the M10 a1 and a2 domains.
By contrast, M10 residues that point into the groove show less
variability (Figure 4A). This pattern of variability is opposite
to the pattern in classical MHC class I molecules, in which
residues that display the greatest sequence variability point
towards the peptide-binding groove, resulting in allelic
specificity for binding peptides, whereas residues on the tops
of the a1 and a2 domain helices are more conserved [43,44]
(Figure 4B).
to the low thermal stability of M10.5, the peptide-filled form
of the H-2Kd heavy chain melts with a Tm of approximately 57
8C [21] (Figure 5B) and FcRn melts with a Tm of approximately 62 8C at pH 6 [47]. The low thermal stability of
purified M10.5 suggests that a ligand or binding partner not
present in the purified preparation is necessary to stabilize
the native conformation at 37 8C in vivo.
M10.5 Is Thermally Unstable
Classical class I MHC molecules and UL18 are thermally
unstable in the absence of bound peptide [21,45,46]. In
contrast, non-peptide binding class I MHC homologs, such as
FcRn and the hemochromatosis protein HFE, do not show
reduced thermal stability, presumably due to structural
rearrangements that close the counterparts of their peptide-binding grooves [32,33,47]. The M10.5 groove is open,
but apparently unoccupied, in the crystal structure, raising
the question of whether M10.5 is stable in the absence of a
groove occupant.
To determine the thermal stability of M10.5, we monitored
heat-induced unfolding by recording the circular dichroism
(CD) signal at 223 nm as a function of increasing temperature. Two unfolding transitions, an upward-sloping transition with a Tm of 43 8C and a downward-sloping transition
with a Tm of 64 8C, are evident in the melting curve of insect
cell-derived M10.5 (Figure 5A). The M10.5 melting curve and
derived Tm values are similar to previously reported results
derived from an empty form of the mouse class I MHC
molecule H-2Kd complexed with human b2m [21,46]. In these
studies, the first transition (Tm ¼ 43–45 8C) represented the
unfolding of the empty H-2Kd heavy chain, and the second
transition (Tm ¼ 64 8C) represented the independent
unfolding of b2m subsequent to heavy chain denaturation
[21,46] (Figure 5B). By analogy, we interpret the first and
second transitions in the M10.5 melting curve as resulting
from M10.5 and b2m denaturations, respectively. In contrast
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M10.5 Does Not Associate with Endogenous Peptides
The empty groove in the M10.5 structure could result from
expression in invertebrate cells, which do not possess the
machinery to load peptides into class I MHC molecules
[48,49]. When classical class I MHC molecules are expressed in
vertebrate cells or purified from native sources, mixtures of
short peptides derived from cytoplasmic proteins can be
eluted [50]. Sequence analyses of eluted peptides revealed
that any given class I allele can bind a wide variety of short (8to 10-mer) peptides that conform to a particular allelespecific peptide-binding motif involving preferred residues at
the peptide C-terminus and an internal position (usually
position 2 or 5/6) [50]. To determine whether M10.5 binds
endogenous or exogenous peptides with these or other
characteristics, we expressed the M10.5 ectodomain in CHO
cells, mammalian cells that support peptide loading, and
examined acid eluates from purified M10.5 using methods
previously used to identify peptides eluted from class I MHC
molecules [51].
Purified M10.5-b2m heterodimers expressed in CHO and
in insect cells were treated with acetic acid to dissociate
potential peptide material. Insect cell-derived M10.5 was
analyzed before and after incubation with a mixture of short
(8- to 9-mer) peptides. As it would be impossible to test all
possible 8- to 9-mer peptides, we prepared a mixture of
peptides including those reported to activate V2R-expressing
neurons [19] in order to see if any of these class I MHC1442
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Crystal Structure of the MHC Ib Molecule M10.5
Table 3. Amino Acid Yields from Acid Eluates
Cycle Number
UL18
FcRn
M10.5 (CHO)
M10.5 (Insect)
M10.5 þPeptide (Insect)
1
2
3
4
5
6
7
8
9
10
11
15.22
11.74
8.89
4.99
4.55
4.52
2.46
3.35
1.95
1.20
1.21
14.69
3.23
3.19
4.68
2.01
2.30
1.85
0.92
1.31
0.00
1.57
1.75
0.60
0.39
0.35
0.66
0.54
0.50
0.40
0.38
0.33
0.49
4.93
1.07
1.15
0.23
1.49
0.51
0.79
0.23
0.65
0.55
0.38
8.10
2.65
0.41
0.74
1.14
0.31
0.95
0.55
0.55
0.84
1.01
Total yield (pmols) of amino acids for each Edman degradation sequencing cycle for acid eluates. Only cycles that exhibited an increase in signal from the previous cycle were included in the total yield and the amino acids threonine and
aspartate were removed due to a large signal from sample contaminants. Acid eluates were derived from equivalent amounts of UL18, FcRn, CHO cell-derived M10.5, insect cell-derived M10.5, and insect cell-derived M10.5 to which a 20-fold
molar excess of the H-2Kd–binding peptide SYIPSAEKI [73,74] was added. Peptide-incubated M10.5 was separated from unbound peptide by gel-filtration chromatography prior to the acid elution procedure. The peptide incubation procedure
was repeated using mixtures of six or 13 MHC–binding peptides (see Materials and Methods) with similar results (i.e., no detectable peptides), as assayed by N-terminal sequencing (for the experiment involving six peptides) or mass
spectrometry (for the experiment involving 13 peptides) (data not shown). MALDI-TOF analysis did not indicate the presence of N-terminally blocked peptides or other small molecules in the M10.5 or FcRn samples (data not shown).
DOI: 10.1371/journal.pbio.0030257.t003
computer modeling to evaluate which potential ligands can
fit into the M10.5 groove.
We first consider whether the M10.5 groove can accommodate a class I MHC–binding peptide by superimposing a
polyalanine version of an H-2Dd–binding peptide [24] or a
Qa-2–binding peptide [22] on the M10.5 structure (see Figure
2C). The H-2Dd–binding peptide fits into most regions of the
M10.5 groove, but clashes with three residues in the A and B
pockets (see Table 2). The Qa-2 peptide adopts a sharper
upward bend near residue P2 (data not shown), and
consequently, two of these clashes (Arg9 and Phe99) do not
occur. The third clash involves Arg167 with the N-terminus of
the peptide, but arginine sidechains are often flexible and
might be able to move out of the way of the N-terminus of a
bound peptide. Several residues in the vicinity of Arg167 have
smaller sidechains in M10.5 than in H-2Dd, which could open
up additional space for alternative conformations of a
peptide terminus. These include Thr7 (Tyr7 in H-2Dd) and
Cys171 (Tyr171 in H-2Dd), as well as Arg167 (Trp167 in H2Dd) if its sidechain can adopt an alternate conformation. We
conclude that the M10.5 groove can accommodate a peptide
that adopts a class I MHC–binding conformation, but that
differences between the A and F pocket regions of M10.5 and
classical class I MHC molecules (see Figure 2B) would require
a peptide bound in the M10.5 groove to be anchored
differently than a class I MHC–binding peptide.
We next considered whether longer peptides or an
extended region of a protein could fit into the M10.5 groove
by extending the polyalanine version of the H-2Dd–binding
peptide by four alanine residues at its C-terminus. The
resulting peptide was fit into the M10.5 groove with the extra
residues extending out of the F pocket side of the groove
(Figure 2C). The relatively open F pocket end of the M10.5
groove is able to accommodate the extra residues, suggesting
that M10 proteins could bind extended regions of other
proteins. Candidate M10–binding proteins include the V2Rs
and/or the major urinary proteins (MUPs), which are thought
to deliver small-molecule compounds to chemosensory
receptor neurons [1,12]. Although the nature of the interaction is unknown, biochemical data demonstrate an association between M10 and V2R proteins [8], thus the open M10.5
binding peptides bind to M10.5. We used soluble versions of
two other b2m-binding class I MHC–like proteins expressed
in CHO cells as controls: UL18, a viral class I MHC homolog
that binds endogenous peptides resembling class I MHC–
binding peptides [52], and FcRn, a class I MHC homolog that
does not bind peptides or contain a non-peptidic groove
occupant [32,47].
Low molecular weight acid eluates derived from M10.5 and
the control proteins were sequenced by Edman degradation
and analyzed by MALDI-TOF (matrix-assisted laser desorption ionization time-of-flight) mass spectrometry. Acid
eluates from CHO–derived M10.5 and both peptide-treated
and untreated insect cell-derived M10.5 resemble eluates
from FcRn, the non–peptide-binding class I homolog, rather
than eluates from UL18, a peptide-binding protein (Table 3).
With the exception of cycle 1, which typically shows a high
background for FcRn and other proteins that do not bind
peptides [52], the total yield of the amino acids from each
cycle of pool sequencing of the M10.5 acid eluates remains
nearly constant and not significantly above background. By
contrast, the UL18 acid eluate shows the presence of a
mixture of peptides with characteristics similar to those
previously reported [52]. MALDI-TOF analysis of the acid
eluates indicated molecules with masses consistent with
peptides in the UL18 sample, but not in the negative control
or M10.5 samples (data not shown). Thus M10.5 does not
appear to bind any form of peptide, including N-terminally
blocked peptides, or other small molecule ligand when
expressed in CHO or insect cells. In addition, purified
M10.5 does not bind any of a collection of class I MHC–
binding peptides, as would have been expected if M10
proteins were the molecules responsible for binding the class
I MHC–binding peptides reported to activate VNO neurons
[19].
Computer Modeling of Potential M10.5 Groove Occupants
Given the open character of the M10.5 groove and the
thermal instability of M10.5-b2m heterodimers, we believe
that the groove is likely to be a binding site for a peptide,
protein, or a non-peptidic small molecule ligand. Here we use
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Crystal Structure of the MHC Ib Molecule M10.5
I proteins, which bind endogenous peptides when expressed
in CHO cells [21], or that it does not bind peptides at all. If
M10.5 does bind peptides, changes in the M10 A and F
pockets from their counterparts in class I MHC grooves (see
Figure 2B) suggest that M10.5 and other M10 molecules
cannot bind the same sort of peptides that are bound by class
I MHC molecules. Thus it is unlikely that the class I MHC–
binding peptides reported to activate VNO neurons [19]
exert their effects by binding to M10 family members,
consistent with the observation that purified M10.5 showed
no detectable binding to peptides used in that study.
Although the ectodomains of classical class I MHC
molecules can fold in the absence of peptide [21,58], and
full-length empty class I proteins can reach the cell surface,
they are thermally unstable and are rapidly degraded unless
an appropriate binding peptide is added exogenously or the
cells are grown at 26 8C [45]. Heat-induced unfolding of
recombinant M10.5 reveals a thermal stability similar to
empty, rather than peptide-filled, class I MHC molecules
(Figure 5A and 5B). It has been assumed that the low thermal
stability of empty class I MHC molecules contributes to the
apparent inability of empty class I molecules to crystallize
(unpublished data). Indeed, the results of molecular dynamics
simulations have been used to predict that empty a1–a2
platforms do not adopt a single defined conformation [59].
However, in the case of the empty M10.5 a1–a2 platform, we
are able to generate moderately well-ordered crystals, and we
see a single conformation for the five copies of M10.5 in the
crystallographic asymmetric unit (Figure S1). The empty
platforms may be stabilized somewhat by crystal contacts, but
different crystal contacts for the five M10.5 molecules do not
produce different conformations of the a1–a2 platform, as
evidenced by relatively low temperature factors for a1–a2
residues (see Materials and Methods).
The low thermal stability of empty recombinant M10.5
suggests that the groove is normally occupied to stabilize the
protein at 37 8C. Although our results cannot be used to
identify a physiologically relevant groove occupant, the
structural and biochemical results can be used to determine
which types of ligands are unlikely. Our analysis of the M10.5
structure and the fact that endogenous peptides were not
found associated with M10.5 expressed in CHO cells (Table 3)
do not support a model in which M10 proteins are stabilized
by class I MHC–binding peptides from either endogenous or
exogenous sources. In addition, the M10.5 groove does not
have the largely hydrophobic character that would be
expected if it were a binding site for hydrophobic pheromones serving as chemical cues in urine, and we observed no
detectable binding signals when fractionated mouse urine
was injected over purified M10.5 in a surface plasmon
resonance-based binding assay (data not shown, see Materials
and Methods).
A clue as to the possible nature of an M10 groove occupant
comes from the different patterns of variability within the
grooves of M10 and classical class I MHC proteins (Figure 4),
which likely reflect the different functional roles of vomeronasal versus classical MHC molecules. Variability within the
grooves of classical class I MHC molecules, which mainly
involves inward-pointing groove residues [43], creates different peptide-binding preferences such that different alleles
present different types of peptides that conform to allelespecific peptide-binding motifs [35]. If the grooves of M10
groove may form a binding site for a piece of a V2R. MUPs,
however, are not known to associate with M10 proteins, and a
surface plasmon resonance-based binding experiment did
not reveal an interaction between immobilized M10.5 and
MUPs in fractionated mouse urine (data not shown; see
Materials and Methods).
It is also possible that pheromones bind within the M10
groove and either are presented directly to V2R receptors or
initiate a conformational change within the M10 molecule
that results in the activation of the receptor. Because there
are no known V2R–activating ligands, we examined pheromones known to activate V1R receptors including 2heptanone, a- or b-farnesene, and 2,5-dimethylpyrazine [53].
Although these molecules fit easily within the M10.5 groove
(data not shown), the hydrophobic nature of most pheromones is not complementary to the charged character of the
groove (see Figure 2A), which is much larger than a single
pheromone molecule. Smaller pheromones might associate
with residues within the relatively hydrophobic M10.5 D
pocket (Figure 2A), however, the rest of the groove would
remain unoccupied and presumably still unstable, and it is
not obvious how such an interaction would lead to activation
of a V2R if it occurs through a conformational change in an
M10 groove.
Discussion
The M10 family of murine class Ib MHC molecules are
expressed exclusively in the VNO and appear to act as
chaperones to facilitate cell surface expression of the V2R
class of pheromone receptors [1,8]. Although the role of M10
molecules in V2R signaling is unclear, direct homologs of
proteins typically associated with immunogenic identity such
as MHC proteins provide attractive candidates to mediate
MHC–disassortative mating preferences [17] and pregnancyblock phenomena [18]. Here we present a biochemical and
structural analysis of M10.5, a representative M10 molecule,
aimed at providing new insights into the function of M10
proteins and their association with V2Rs. As M10 proteins are
related by greater than 60% sequence identity [8,12], above
the approximately 30% sequence identity threshold suggesting similar three-dimensional structures [54], the results
obtained for M10.5 are relevant to other M10 proteins.
Proteins with an MHC fold generally have open, occupied
grooves, as in classical MHC proteins, other class Ib proteins,
CD1 [30], Zn-a2-glycoprotein [28], and the protein C receptor
[55], or closed, unoccupied grooves, as in FcRn [32], HFE [33],
MIC-A [34], Rae-1b [56], and T22 [57]. Thus it is surprising
that the M10.5 groove is open, but apparently unoccupied, in
the crystal structure. Consistent with the lack of defined extra
electron density for a groove occupant, analyses of acid
eluates derived from M10.5 expressed in insect cells and in
CHO cells failed to reveal peptidic or non-peptidic material
(Table 3). In addition, M10.5 did not bind MHC–binding
peptides, including peptides reported to activate VNO
neurons expressing M10 family members [19]. These results
do not rule out a peptide-binding role for M10 proteins
because the peptides used for these experiments may not
have been optimal for binding to M10.5. However, the fact
that M10.5 does not associate with any endogenous peptides
when expressed in CHO cells suggests that it either has more
stringent criteria for peptide binding than conventional class
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Crystal Structure of the MHC Ib Molecule M10.5
subcloned into the BamHI site of the dicistronic baculovirus transfer
vector pAcUW31 (BD Biosciences Clontech, Mountain View, California, United States). The ectodomain was truncated after residue
Gly299 (Gly275 using numbering corresponding to mature class I
MHC heavy chains). cDNA encoding human b2m plus its hydrophobic leader sequence was inserted into the BglII site of the transfer
vector. Recombinant baculovirus was generated by co-transfection of
the transfer vector with linearized viral DNA (Baculogold; BD
Biosciences Clontech) and supernatants were harvested from
baculovirus-infected Tn5 (High Five) insect cells. Cell supernatants
were buffer exchanged into 20 mM Tris (pH 7.4)/150 mM NaCl, and
concentrated to 1 liter using an Amicon RA2000 tangential-flow
concentrator (Amicon, Beverly, Massachusetts, United States) with a
10 kDa cutoff membrane (Pall Corporation, East Hills, New York,
United States). The resulting solution was adjusted to 50 mM Tris (pH
7.4)/300 mM NaCl/10 mM imidazole/10% glycerol, and loaded onto an
8-ml Ni-NTA column (Qiagen, Valencia, California, United States)
equilibrated in 50 mM Tris (pH 7.4)/300 mM NaCl/10% glycerol. The
column was washed with a similar buffer containing 40 mM imidazole
and the bound M10.5 eluted in 250 mM imidazole. M10.5-containing
fractions were pooled, concentrated to 2 ml, and loaded onto a 16/60
Superdex 75 column (Amersham Biosciences Corp., Piscataway, New
Jersey, United States) equilibrated in 20 mM Tris (pH 7.4)/150 mM
NaCl/1 mM EDTA/1 mM b-mercaptoethanol. SDS-PAGE analysis of
the major peak on the gel filtration column revealed two bands
migrating with apparent molecular masses of 37 kDa (expected mass
32.6 kDa þ carbohydrate), corresponding to the M10.5 ectodomain,
and 12 kDa (expected mass 11.7 kDa), corresponding to b2m. The
M10.5-b2m peak eluted at a position corresponding to a protein of
approximately 47 kDa, suggesting that the protein is monomeric (i.e.,
a single heterodimer). Fractions containing M10.5 were pooled
resulting in a yield of approximately 1.5 mg per liter of insect cell
supernatant.
N-terminal sequencing of the M10.5 heavy chain was accomplished
by first blotting gel-run protein to a PVDF membrane (polyvinylidene
fluoride, Millipore, Billerica, Massachusetts, United States), followed
by sequencing using a 492 cLC Procise protein micro-sequencer
(Applied Biosystems, Foster City, California, United States). The
sequence obtained, SHWLKT, corresponds to the processed mature
form of M10.5. In our numbering system, the first amino acid of the
processed M10.5 heavy chain is denoted as residue 2 to correspond
with the numbering of the processed forms of classical class I MHC
molecules.
A vector for expression of M10.5 in CHO cells was constructed by
subcloning the analogous region of M10.5 into a derivative of pBJ5GS, which carries the glutamine synthetase gene as a selectable
marker and means of gene amplification in the presence of
methionine sulfoximine [62]. The M10.5 expression vector was
transfected into CHO cells together with a human b2m expression
vector as described [33]. Selection, amplification, maintenance of
methionine sulfoximine-resistant cells, and identification of M10.5expressing cells were done as described [46]. M10.5-b2m heterodimers were purified from CHO cell supernatants as described for
the insect cell-derived protein.
Crystallization and data collection. Crystals (space group P212121; a
¼ 124.11 Å, b ¼ 124.71 Å, c ¼ 149.37 Å; five molecules per asymmetric
unit) were grown at room temperature utilizing the hanging drop
method by combining 1 ll of protein solution (approximately 8 mg/
ml of insect cell-derived M10.5) with 1 ll of precipitant containing 0.1
M imidazole (pH 8.0)/20% PEG 1000/0.2 M calcium acetate. Crystals
were cryopreserved in liquid nitrogen after soaking in mother liquor
containing 10% glycerol as a cryoprotectant. Native datasets were
collected at 100 K at beamline 12.3.1 at the Advanced Light Source
(Berkeley, California, United States) and beamline 9–2 at the Stanford
Synchrotron Radiation Laboratory (Stanford, California, United
States). The data were indexed and scaled using the HKL suite of
programs (HKL Research, Charlottesville, Virginia, United States)
(see Table 1). The statistics reported in Table 1 refer to a single
merged native dataset obtained by including frames from both native
datasets in the scaling procedure.
Structure solution and refinement. A protein–protein BLAST
search indicated that Qa-2 and H-2Dd are the closest-related proteins
in sequence to M10.5 for which crystal structures are available.
Molecular replacement was carried out using the CCP4 program
AMORE [63,64] with an all atom version of Qa-2 (not including the
bound peptide) as a search model. Molecular replacement was not
successful using each dataset individually, but a solution was obtained
when the data were merged into a single native dataset (see Table 1).
Five molecules were located in the asymmetric unit giving an R-factor
of 47.9% after rigid-body refinement.
proteins are occupied, the relative conservation of residues
that point toward the M10 groove suggests that different M10
proteins bind a more limited set of ligands than the ligands of
different class I MHC alleles. The greater variability in the
upward-pointing residues on the M10 helices (Figure 4A)
suggests allele-specific interactions with other proteins,
consistent with the suggestion of specificity in binding
between different V2R and M10 proteins [8]. The variability
analysis combined with the M10.5 structure suggests a
potential interaction site: The disordered six-residue loop
within the M10.5 a2 domain (residues 145–150) contains
three highly variable residues that could be involved in an
interaction with a V2R. As a precedent for a disordered
region of an MHC-like structure being at a receptor binding
site, a portion of the a2 domain helix that is disordered in the
structure of MIC-A (residues 152 to 161, corresponding to
approximately the same M10.5 residues) [34] becomes
ordered in a structure of MIC-A bound to the NKG2D
receptor [60].
The disordered region of the M10.5 a2 domain helix may
also be related to a need for flexibility within the F pocket
region of the M10 groove, which could allow binding of larger
ligands than the 8- to 10-mer peptides bound in the grooves
of classical class I MHC molecules. Although the M10.5 groove
is about the same size as the grooves of classical class I MHC
molecules (approximately 730 Å2 versus approximately 760
Å2), and the A pocket side of both types of grooves is closed,
the F pocket side of the M10.5 groove is more open than the
counterpart region of a class I molecule (see Figure 2). The
closed ends of class I MHC grooves result in the preference
for binding short (8- to 10-mer) peptides that do not extend
out of either end of the groove [35]. The open F pocket end of
the M10.5 groove may allow it to bind larger ligands, perhaps
even an extended region of an intact protein, such as a V2R.
As a precedent for this type of interaction, the open grooves
of class II MHC molecules bind to an extended region with
the invariant chain protein during transit through the ER and
Golgi (reviewed in [61]). In this case, the invariant chain not
only occludes the class II MHC groove to prevent association
with ER peptides, it also serves as a chaperone to direct class
II proteins to acidic compartments where they acquire
peptides derived from exogenous proteins. A related situation could occur for M10 proteins, such that an extended
region within the ectodomain of a V2R would bind within the
groove of an M10 protein during transit through the ER and
Golgi. An interaction in which an extended loop from a V2R
protein binds to an M10 groove would explain why peptides
or other small molecule ligands were not found in recombinant M10.5 molecules expressed in the absence of V2R
proteins and why the empty recombinant molecules are
thermally unstable. In this hypothesized scenario, newly
synthesized M10 and V2R proteins would be stabilized
through mutual interactions with a V2R loop in the M10
groove, enabling the M10 to escort the V2R to the cell surface,
rationalizing the observation that M10 proteins are required
for cell surface expression of V2Rs [8].
Materials and Methods
M10.5 expression and purification. A construct encoding soluble
M10.5 (corresponding to the ectodomain with the preceding hydrophobic leader sequence and an upstream insect Kozak sequence
(CCTATAAAT) plus a C-terminal Factor Xa site and a 6xHis tag) was
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Crystal Structure of the MHC Ib Molecule M10.5
The model was refined using all reflections to 3.0 Å, but as the data
are only approximately 60% complete between 3.1 Å and 3.0 Å, the
effective resolution of the structure is approximately 3.2 Å. Test set
reflections (5% of total) were picked using the thin shell method in
DATAMAN [65] to reduce the influence of NCS correlations on Rfactor calculations. To reduce the possibility of model bias, initial
averaged maps were generated using the Qa-2 structure (open
groove) or the FcRn structure (closed groove) to generate model
structure factors. Maps produced by both methods indicated an open
M10.5 groove. The M10.5 model was built using the program O [66]
into real space–averaged, annealed composite omit electron-density
maps in which 5% of the molecule was omitted at a time. Simulated
annealing and grouped B-factor refinement with 5-fold NCS
constraints was carried out using CNS [67]. Once the Rfree value
stopped improving with successive cycles of refinement, the NCS
constraints were relaxed to NCS restraints using a weight of 300 kcal/
molÅ2. Parts of the model that significantly deviated between NCSrelated molecules or that formed packing contacts were removed
from the NCS restraints. The model stereochemistry was checked
after each round of refinement using PROCHECK [68] and
WHAT_CHECK [69]. The model (Rcryst ¼ 26.6%; Rfree ¼ 30.3%)
includes 264 out of 274 residues in the M10.5 heavy chain and all 99
residues of b2m (Table 1). Residues 145–150 and 195–198 are not seen
in the electron-density map, and 36 sidechains in the molecule 1
M10.5 heavy chain and 13 sidechains in b2m are disordered and were
modeled as alanines. The average main chain temperature factors per
domain for molecule 1 are 48.3 Å2 (a1–a2), 68.2 Å2 (a3), and 44.4
Å2 (b2m). The temperature factors are the highest in the distal half of
the a3 domain, correlating with this region having less continuous
electron density than other regions of the maps.
Groove surface area calculations were performed as previously
described [30,33] using the GRASP program [42]. Alignments of the
M10.5 a1- a2 domains with other a1–a2 platforms were carried out
with the CCP4 program Lsqkab [63] using platform b-sheet residues
3–13, 21–28, 34–37, 46–47, 93–103, 111–118, 122–126, and 133–135.
Figures were produced using Molscript [70], Raster3D [71], and
PyMOL [72].
Thermal stability analyses. An AVIV 62A DS CD spectrometer with
a thermoelectric cell holder and a cuvette with a 1-mm path length
was used to monitor heat-induced unfolding of insect cell-derived
M10.5 using samples containing 15 lM protein in 50 mM phosphate
buffer. In one experiment, the protein solution also contained 20%
polyethylene glycol 1000. The CD signal was monitored at 223 nm
while the temperature was increased from 1 to 1008C in 1 degree
increments with an equilibration time of 2 min and an averaging time
of 30 s. Tms were determined by estimating the half-point of the
ellipticity change between the pure native and pure denatured states.
Acid elutions and peptide sequencing. Samples of purified M10.5
expressed in CHO and insect cells were analyzed for the presence of
bound peptides as previously described [46]. In these experiments,
FcRn (or HFE in an independent experiment; data not shown) served
as a negative control since it had been previously established by
biochemical and crystallographic methods that FcRn and HFE do not
associate with endogenous peptides [32,33,47]. UL18 served as the
positive control, because it associates with endogenous peptides when
expressed in CHO cells [46,52]. A 20-fold molar excess of the H-2Kd–
binding peptide SYIPSAEKI [73,74] was added to insect cell M10.5
followed by gel-filtration chromatography to remove unbound
peptide. This was repeated twice using a mixture of peptides, each
at a 10-fold molar excess compared to M10.5. One mixture contained
13 peptides, including the following peptides used in a study
reporting peptide-induced activation of VNO neurons [19]: AAPDNRETF (binds to H-2Db), AAPDARETA (mutated form of first peptide),
ASNENMETM (binds to H-2Db), FAPGNYPAL (binds to H-2Db),
SYFPEITHI (binds to H-2Kd), SAFPEITHA (mutated form of
preceding peptide), SYIPSAEKI (binds to H-2Kd), SFVDTRTLL (binds
to H-2Kd), plus five other peptides: ALPHAILRL (binds to UL18) [52],
TYCRTRALV (modified from an H-2Kd–binding peptide) [51,74],
RGYLYQGL (binds to H-2Kb) [75], FAPGVFPYM (binds to H-2Db)
[76], and ovalbumin-derived SIINFEKL (binds to H-2Kb) [77]. The
other mixture contained six of the above peptides. Acid elutions and
sequencing were performed by established methods [51,78,79].
Briefly, 250 lg of CHO-derived and insect cell-derived soluble
M10.5 (peptide-treated and untreated), UL18, and FcRn were
concentrated to 100 ll in a Centricon 3 kDa cutoff centrifugal
concentrator (Amicon). After addition of 1 ml of 50 mM ammonium
acetate (pH 7.5), the proteins were again concentrated to 100 ll. This
washing step was repeated once, followed by the addition of 1 ml of
10% acetic acid. The samples were heated to 70 8C for 15 min, and
the solutions concentrated to 100 ll. This elution step was repeated
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once and the resulting 2 ml of filtrate was concentrated by
evaporation to 50 ll. Automated Edman degradation was performed
on 10 ll using a 492 cLC Procise protein micro-sequencer (Applied
Biosystems). Analysis of acid eluates was also performed using a
PerSeptive Biosystems Voyager Elite MALDI-TOF mass spectrometer
(PerSeptive Biosystems, Framingham, Massachusetts, United States)
with delayed extraction and a high sensitivity linear detector.
Surface plasmon resonance binding assay. A Biacore 2000
biosensor system (Pharmacia-LKB Biotechnology, Uppsala, Sweden)
was used to monitor interactions between M10.5 and potential
ligands in mouse urine. Purified insect cell-derived M10.5 was
coupled to two flow cells of a CM5 biosensor chip (Pharmacia-LKB
Biotechnology) to coupling densities of 1600 and 4900 resonance
units using standard amine coupling chemistry. Purified FcRn was
coupled to a third flow cell to 1600 resonance units as a negative
control, and a fourth flow cell was mock coupled. Urine was freshly
collected from adult male and female C57BL/6 mice, and samples
were frozen immediately. Male mouse urine (300–500 ll) was desalted
through a G25 protein desalting spin column (Pierce Biotechnology,
Rockford, Illinois, United States) prior to loading directly onto a
RESOURCE Q (1 ml) high-performance ion exchange column
(Amersham Biosciences). The column was then washed in 5 ml of
buffer A (10 mM phosphate buffer [pH 7.0]), followed by elution in 10
ml of a linear gradient of buffer A to buffer B (10 mM phosphate
buffer [pH 7.0]/300 mM NaCl) using a Vision Workstation HPLC
system (Applied Biosystems). The concentration of MUPs in the
HPLC fractions were estimated to be approximately 0.5 mg/ml based
on Coomassie-stained SDS-PAGE gels and absorbance at 280 nm.
Unfractionated urine contained approximately 100-fold higher
concentrations of MUPs based on the gel staining. Whole and
fractionated urine samples were diluted 10-fold into a buffer
containing 50 mM HEPES (pH 7.4)/150 mM NaCl and injected at
room temperature over the M10.5-, FcRn-, and mock-coupled flow
cells. None of the samples showed any measurable responses aside
from nonspecific refractive index changes, and no significant differences were observed between M10.5- and FcRn-coupled cells (data
not shown). Although these results rule out binding between M10.5
and protein components in urine (e.g., MUPs, which are approximately 19 kDa), detection of molecules smaller than 1 kDa using a
Biacore 2000 is problematic due to noise and buffer effects. As a
positive control for the integrity of the coupled M10.5 proteins, a
rabbit polyclonal antiserum raised against insect cell-purified M10.5
was injected over all four flow cells giving a high-affinity response
only on the M10.5-coupled cells (data not shown).
Supporting Information
Figure S1. Groove Fo-Fc Electron-Density Maps for NCS-Related
Molecules
(A) Sigma-A–weighted Fo-Fc maps contoured at 3.0 r showing
electron density within the a1–a2 domain. Electron density is shown
for each of the five molecules in the asymmetric unit. There is no
density indicative of bound peptide(s) or another type of groove
occupant, except for a small peak in 5-fold averaged maps, which may
result from approximately six carbons of a weakly interacting
polyethylene glycol molecule in a location similar to that seen for a
hexaethylene glycol molecule in the groove of Zn-a2-glycoprotein
[27], which, like M10.5, was crystallized in the presence of polyethylene glycol. The peak could account for about half of a
hexaethylene glycol molecule, but by contrast to the more ordered
and stronger polyethylene glycol density in the Zn-a2-glycoprotein
groove, the extra electron density within the M10.5 groove is weak
and discontinuous. Thus if M10.5 does bind polyethylene glycol, it
does not bind it in a single, ordered conformation, as observed in the
structure of Zn-a2-glycoprotein [27], and probably does so to
approximate a higher-affinity natural ligand.
(B) Real space–averaged Fo-Fc map using the M10.5 model refined
with 5-fold NCS constraints.
Found at DOI: 10.1371/journal.pbio.0030257.sg001 (2.0 MB JPG).
Accession Numbers
Uniprot accession numbers (http://www.pir.uniprot.org) for proteins
discussed in this paper are as follows: b2m, human (P61769), b2m,
mouse (Q91XJ8), C receptor (Q9UNN8), CD1 (P11609), CD8 (P01731),
FcRn (P13599), GABAB (Q9WV18), Gai2 (P08752), Gao (P18872), H2Dd (P01900), H-2Kb (P01901), H-2Kd (P01902), H2-M3 (Q31093), HFE
(Q6B0J5), HLA-E (P13747), M10.5 (Q85ZW7), MIC-A (Q5C9P8),
1446
August 2005 | Volume 3 | Issue 8 | e257
Crystal Structure of the MHC Ib Molecule M10.5
NKG2D receptor (P26718), Qa-2 (P79567), Rae-1b (O08603), T22
(Q9BCZ1), TAP1 (Q62427), TAP2 (P36371), UL18 (P08560), and Zna2-glycoprotein (P25311).
The Research Collaboratory for Structural Bioinformatics (RCSB)
Protein Data Bank accession number (http://www.rcsb.org/pdb/) for
the M10.5 structure is 1ZS8. The accession numbers for the other
proteins discussed in this paper are as follows: FcRn (3FRU), H-2Dd
(1BII), and Qa-2 (1K8D).
Protein sequencing analyses carried out by the Protein/Peptide
Microanalytical Lab were supported by the Beckman Institute at
the California Institute of Technology (Caltech). We thank Kyle
Lassila and Heidi Privett for help with CD measurements, Tony
Giannetti and Anthony West for help with Biacore experiments,
Peter Snow, Inderjit Nangiana, and Cynthia Jones at the Caltech
Protein Expression Center for assisting with insect cell expression,
Fabio Papes for providing fractionated urine samples, the Ralph
M.Parsons Foundation for computational support, and members of
the Bjorkman laboratory for critical reading of the manuscript. This
work was supported by a Rosalind Alcott Post-doctoral Fellowship
administered by Caltech (R.O.), the Howard Hughes Medical Institute
(C.D. and P.J.B.), and the NIH/NIDCD (R01 DC003903 (C.D.)).
The Advanced Light Source is supported by the Director, Office of
Science, Office of Basic Energy Sciences, Materials Sciences Division,
of the U.S. Department of Energy under Contract No. DE-AC0376SF00098 at Lawrence Berkeley National Laboratory. Portions of
this research were carried out at the Stanford Synchrotron Radiation
Laboratory, a national user facility operated by Stanford University
on behalf of the U.S. Department of Energy, Office of Basic Energy
Sciences.
Competing interests. The authors have declared that no competing
interests exist.
Author contributions. RAO and PJB conceived and designed the
experiments. RAO and KEHT performed the experiments. RAO,
KEHT, CD, and PJB analyzed the data. CD and PJB contributed
reagents/materials/analysis tools. RAO, CD, and PJB wrote the paper.&
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